P849: DEPLETION OF DIS3 IN MULTIPLE MYELOMA LEADS TO PERTURBATION IN RNA METABOLISM, CELL CYCLE PROGRESSION AND MITOTIC CHECKPOINT.
Authors: V. Favasuli, I. Silvestris, K. Todoerti, D. Ronchetti, S. Erratico, D. giannandrea, Y. Torrente, R. Chiaramonte, S. Fabbris, N. Bolli, L. Baldini, A. Neri, E. Taiana
Journal: HemaSphere (2022)

Background: MM is a genetically heterogeneous neoplasm in which the co-occurrency of multiple genomic events play a crucial role in tumor development, progression and drug resistance. DIS3 gene maps at 13q22 and encodes for a highly conserved ribonuclease involved in the RNA processing, quality control and degradation. Deletion of DIS3 gene is a common characteristic of MM patients (approximately 50% of cases); moreover, DIS3 mutations occur in about 11% of MM cases and are frequently (70%) associated with loss of the gene. DIS3 mutations along with del(13q) have been recently described by us as associated with a strong negative effect on clinical outcome. Aims: The purpose of this study is to shed light on the biological role and impact of DIS3 transcript in MM. Methods: LNA gapmeRs were used to silence DIS3 in HMCLs by gymnotic delivery in four human myeloma cell lines, two of which showing a monoallelic del(13q). qRT-PCR has been used to evaluate DIS3 silencing efficiency. Cell viability and cellular growth was evaluated by viable counts and by Cell Titer Glo assay. Modulation of cell cycle phases distribution was assessed by flow cytometry. Cell cycle synchronization was obtained using SynchroSet kit. Transcriptional analysis was performed using Affymetrix microarray. Protein expression was evaluated by Western Blot. Mitotic spindle organization was analyzed by immunofluorescence (IF). Results:DIS3 silenced HMCLs show a significant decrease of cellular growth right after 4 days from LNA gapmeR delivery. DIS3depleted cells showed a significant perturbation of cell cycle in all the HMCLs tested. Analysis of synchronized HMCLs revealed a more pronounced modulation of all cell cycle phases distribution confirming the involvement of DIS3 in cell cycle regulation. Accordingly, DIS3 silencing lead to an important modulation of cell cycle checkpoint proteins and mitotic spindle checkpoints. Confocal microscopy analysis performed in DIS3 silenced HMCLs revealed the presence of mitotic defects and microtubule organization abnormalities. Transcriptional profile analysis of DIS3-KD samples is associate to a strong modulation of cell cycle regulation and mitotic spindle related pathways, together with the expected, strong modulation of RNA degradation pathway. Summary/Conclusion: Our data indicate that DIS3 silencing in HMCLs leads to a cell cycle perturbation accompanied to an increase of mitotic defects. To the best of our knowledge, this is the first evidence in the context of the pathological role of DIS3 in MM. These results should be considered of interest considering that DIS3 abnormalities are a common feature of MM patients. Moreover, the transcriptional analyses may contribute to identify putative DIS3-related pathways and genes (with coding or non-coding potential) involved in tumorigenic mechanisms in MM and possibly implicated in the therapeutic settings.